1. Field of the Invention
The invention relates to the chemical synthesis of oligonucleotides and to chemical entities and processes useful in such synthesis.
2. Summary of the Related Art
Oligonucleotides have become indispensible tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modem approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23: 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager el al., Biochemistry 27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Antl. Acad. Sci. USA 85: 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
Solid phase synthesis of oligonucleotides by each of the foregoing processes involves the same generalized protocol. Briefly, this approach comprises anchoring the 3xe2x80x2-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Desired internucleoside linkages are formed between the 3xe2x80x2 functional group of the incoming nucleoside and the 5xe2x80x2 hydroxyl group of the 5xe2x80x2-most nucleoside of the nascent, support-bound oligonucleotide.
Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10 xcexcmol to 1 mmol and higher). See Padmapriya et al., Antisense Res. Dev. 4: 185 (1994). Several modifications of the standard phosphoramidite processes have already been reported to facilitate the synthesis (Padmapriya et al., supra; Ravikumar et al., Tetrahedron 50: 9255 (1994); Theisen et al., Nucleosides and Nucleotides 12: 43 (1994); and Iyer et al., Nucleosides and Nucleotides 14: 1349 (1995)) and isolation (Kuijpers et al. Nucl. Acids Res. 18: 5197 (1990); and Reddy et al., Tetrahedron Lett. 35: 4311 (1994)) of oligonucleotides.
The routine synthesis of oligonucleotides is presently carried out using various N-acyl protecting groups for the nucleoside bases, such as isobutyryl (for guanine), and benzoyl for adenine and cytosine. After the synthesis of the oligonucleotides is carried out using either phosphoramidite chemistry or H-phosphonate chemistry, the protecting groups are removed by treatment with ammonia at 55-60xc2x0 C. for 5-10 hours. Using these protecting groups, PO oligonucleotides and other modified oligonucleotides can be synthesized. However, it would be greatly advantageous to be able to carry out such synthesis more rapidly, which would be possible if the time required for removal of the protecting groups could be reduced.
In addition, when currently available deprotection conditions are used, in certain instances where modified oligonucleotides are functionalized with base-sensitive groups, the functionalities often get removed while the deprotection is being carried out. Examples of such base-sensitive modified oligonucleotides include, ribonucleoside-containing oligonucleotides, methylphosphotriester oligonucleotides, phosphoramidates, etc.
One such example is the large-scale synthesis of RNA which is required for the ribozyme-based therapeutic strategies. Such synthesis presents special challenges due to two factors. These are, first, 3xe2x80x2-5xe2x80x2 to 2xe2x80x2-5xe2x80x2 internucleotide chain migration during preparation of nucleoside monomer precursors, during synthesis, and during removal of protecting groups from the RNA, and second, degradation of RNA. Use of classical protecting groups compounds these factors. For successful RNA synthesis, it is essential that the 2xe2x80x2 hydroxyl protecting group remains intact until the final deprotection step and that following its removal, the 2xe2x80x2 hydroxyl group does not attack the vicinal phosphodiester groups and thereby promote cleavage or migration of the internucleotidic linkages.
Another example is that current synthesis procedures allow the synthesis of some, but not all possible oligonucleotide phosphoramidates, because some of these compounds are labile under the highly alkaline conditions required for deprotection of the nucleoside base. Oligonucleotides containing primary phosphoramidate internucleoside linkages, for example, have not previously been possible to synthesize for this reason. In the case of the oligonucleotide phosphoramidates, this inability to synthesize oligonucleotides containing primary phosphoramidate internucleoside linkages has probably slowed their development as optimally useful compounds for molecular biology applications and the antisense therapeutic approach. This is likely because the oligonucleotide phosphoramidates that have been developed all have relatively large chemical substituents in place of one of the nonbridging oxygen atoms on the phosphate backbone, which may lead to steric hindrance in the ability of the oligonucleotide to bind to its target. It would be valuable to have internucleotidic primary phosphoramidate linkages, since incorporation of such non-ionic linkages could result in a reduction in oligonucleotide side effects that are attributable to the polyanionic character of the oligonucleotides. For example, Galbraith et al., Antisense Research and Development 4: 201-206 (1994) disclose complement activation by oligonucleotides. Henry et al., Pharm. Res. 11: PPDM8082 (1994) discloses that oligonucleotides may potentially interfere with blood clotting.
Yet another example is the synthesis of oligonucleotides containing methylphosphonate internucleoside linkages. Various methodologies have been used to synthesize such oligonucleotides. Miller et al., Biochemistry 25: 5092-5095 (1986), discloses an early methodology using a polymer support. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches a more generally applicable phosphoramidite approach using a controlled pore glass (CPG) support. All of the existing approaches, however, are inherently limited by the susceptibility of the methylphosphonate linkage to hydrolysis by base, which precludes the use of the usual deprotection step, which employs prolonged treatment with 28% ammonium hydroxide. Some attempts to deal with this problem have included the use of N-isobutyryl-protected cytidine nucleoside phosphonamidite monomers in conjunction with dAbz and dGiBu monomers, followed by deprotection using initial exposure of the oligomer to 10% ammonium hydroxide in acetonitrile/ethanol at room temperature, then prolonged exposure to ethylenediamine. Also used has been pretreatment of the protected oligonucleotide with hydrazine hydrate in pyridine/acetic acid, followed by prolonged exposure to ethylene diamine/ethanol. Although these approaches have provided somewhat inconvenient answers to certain problems, they have created problems of their own for large scale synthesis of chimeric oligonucleotides, which have segments of different internucleosidic linkages. For example, the dGiBu-methylphosphonamidite monomer is insoluble in acetonitrile, which is the solvent commonly used with most other phosphoramidite monomers. Consequently, prior to each coupling step at which this monomer is added, it is necessary to thoroughly wash the monomer delivery lines and the synthesis column with a solvent in which the dGiBu monomer is soluble, such as anhydrous peroxide-free THF or CH3CN/CH2Cl2, to avoid precipitation of this monomer in the delivery lines or column.
Still another example is synthesis of oligonucleotides containing methyphosphotriester internucleoside linkages. Such oligonucleotides could have many beneficial properties, because the methyl phosphotriester group is nonionic, but is similar in size and molecular shape to the phosphodiester linkage. Such nonionic methyl phosphotriester linkages could result in a reduction in oligonucleotide side effects that are attributable to the polyanionic character of the oligonucleotides.
The art has recognized the desirability of incorporating methyl phosphotriester internucleotide linkages into oligonucleotides and many attempts have been made to make and use such oligonucleotides. However, these attempts have subsequently been discovered to be unsuccessful. Miller et al., J. Am. Chem. Soc. 93: 6657-6665 (1971), discloses alleged methylphosphotriester DNA synthesis by methylation of the phosphate using p-toluenesulphonyl chloride and methanol. Moody et al., Nucl. Acids Res. 17: 4769-4783 (1989), discloses regiospecific inhibition of DNA duplication by oligonucleotides synthesized according to the process of Miller et al. Buck et al., Science 248: 208-212 (1990), discloses that oligonucleotides according to Moody et al. inhibit viral infectivity of HIV-1. However, Buck et al., Science 249: 125-126 (1990), retracts the earlier Buck et al. report and discloses that oligonucleotides synthesized according to this process do not contain methyl phosphotriester internucleotide linkages.
The difficulty in synthesizing oligonucleotides having methyl phosphotriester internucleotide linkages is due to the lability of the methyl ester bond under the oligonucleotide synthesis conditions used in the steps of deprotecting the nucleoside bases and cleaving the oligonucleotides from the solid support. Alul et al., Nucl. Acids Res. 19: 1527-1532 (1991), addressed the problem of cleaving the oligonucleotide from the solid support by introducing an oxalyl-type linker that can be cleaved under conditions that preserve the methyl ester bond. However, the problem of base deprotection was not addressed, so they were only able to synthesize methyl phosphotriester-linked thymidines, which lack an exocyclic amino group and thus do not require deprotection. Kuijpers et al., Nucl. Acids Res. 18: 5197-5205 (1990), attempted to address the deprotection problem by treating the FMOC-protected nucleoside bases for 43 hours in potassium carbonate/methanol. Unfortunately, NMR analysis of their oligonucleotides revealed that considerable demethylation had occurred, resulting oligonucleotides having a mixture of methylphosphotriester and phosphodiester linkages. Similarly, Vinogradov et al., Tetrahedron Lett. 34: 5899-5902 (1993), attempted to solve the problem by using an isopropoxyacetyl group to protect the nucleoside bases, but found that at least 35-40% demethylation still occurred. Most recently, Hayakawa et al., J. Org. Chem. 60: 925-930 (1995), claimed to have synthesized a decamer oligonucleotide containing a single methyl phosphotriester internucleotide linkage. However, NMR data supporting this claim was absent. Moreover, the process utilized costly and toxic palladium, which could contaminate the oligonucleotide product and render it unsuitable for therapeutic applications. In addition, the process was not shown to be able to introduce multiple methylphosphotriester linkages into the oligonucleotide.
In other applications of oligonucleotides, it is desirable to have oligonucleotides still bound to the solid support. Such completely deprotected oligonucleotides still bound to the solid support can be useful in a variety of applications such as those involving isolation of transcription factors and other factors or elements that interact with oligonucleotides. They are also useful for solid-phase PCR, investigation into nucleic acid protein interactions by, for example, NMR, creation and use of combinatorial libraries, screening of nucleic acid libraries, and solid support based hybridization probes (analogous to Southern and Northern blotting protocols). Creating such a support bound, deprotected oligonucleotide would be greatly aided by having a protecting group that could be removed by mild conditions that would not cleave the oligonucleotide from the support.
It would further be desirable to covalently attach certain ligands to oligonucleotides via ester or amide linkages. This would be most advantageously performed when the oligonucleotide is still bound to the solid support because most nucleophilic sites on the oligonucleotide will be blocked by protecting groups and thus will not interfere during ligand conjugation; also because the procedure is simplified by the ability to wash away reagents and solvents in the liquid phase.
These numerous examples clearly demonstrate a need for processes for oligonucleotide synthesis that allow for deprotection of the oligonucleotide under more mild conditions than existing processes. There is further a need for nucleoside synthons having new base protecting groups that are stable under oligonucleotide synthesis conditions, but which can be removed under more rapid and/or more mild conditions than existing protecting groups. Finally, there is a need for oligonucleotides which contain any of a variety of base labile functionalities. An ideal solution to these needs would require a process which allows oligonucleotides to be recovered at higher yields, while simultaneously providing milder conditions.
The invention provides new processes for synthesizing oligonucleotides that allow for deprotection of the oligonucleotide under more rapidly and under more mild conditions than existing processes. The invention further provides a nucleoside base protecting group that is stable under oligonucleotide synthesis conditions, but which can be removed under more mild conditions than existing protecting groups, as well as nucleoside synthons having such base protecting groups. The invention also provides oligonucleotides containing any of a variety of base labile functionalities and methods for using such oligonucleotides.
In a first aspect, the invention provides a novel nucleoside base protecting group having the general structure I: 
wherein n1, n2 and n3 are independently 0-10, wherein a, b, c, d and e are each independently hydrogen, carbon or nitrogen, and wherein the ring structure bearing substituent R3 shown may be aromatic or heterocyclic, wherein the nitrogen displayed is the protected amino moiety of the nucleoside base, wherein R1, R2 and R3 are independently hydrogen, or an alkyl, aryl, aralkyl, ether, hydroxy, nitrile, nitro, ester, carboxyl, or aldehyde group, and wherein dotted lines represent alternative exocyclic or endocyclic double bonds. In a preferred embodiment, a is hydrogen when n1 is 0 and is carbon or nitrogen when n1 is 1-10, b is hydrogen when n1 and n2 are both 0 and is carbon or nitrogen when either or both n1 and n2 are 1-10, c is hydrogen when n2 is 0 and is carbon or nitrogen when n2 is 1-10, and e is hydrogen when n3 is 0 and is carbon or nitrogen when n3 is 1-10. In a particularly preferred embodiment, compound I has n1, n2 and n3 values of 0, and a, b, c, d and e are each hydrogen, and the protecting group takes the form N-pent-4-enoyl, i.e., CH2xe2x95x90CH(CH2)2COxe2x80x94 (II). Compounds I and II protect the nucleoside base amino moieties by forming amide linkages, as in: 
where the nitrogen displayed is the protected amino moiety of the base B.
Base protecting group I and the preferred embodiment II are particularly advantageously used because such protecting group can be removed chemoselectively by treatment with a chemoselective removing agent. Thus, in a second aspect, the invention provides a process for synthesizing oligonucleotides that allows for removal of base protecting groups under more mild conditions than existing processes. This new process comprises sequentially coupling nucleoside synthons having base protecting groups according to the invention to produce a base-protected oligonucleotide, followed by deprotection using a chemoselective removing agent. The process according to the invention can utilize any known or otherwise suitable oligonucleotide synthesis chemistry, including the well known H-phosphonate, phosphoramidite and phosphotriester chemistries.
The use of this new process provides numerous advantages. For example the process""s mild procedure for removing the protecting group without affecting the integrity of other functionalities present in the oligonucleotide makes it possible to prepare novel analogs of oligonucleotides such as ribonucleoside-containing oligonucleotides, alkylphosphotriesters, certain base-sensitive phosphoramidate and other base-sensitive oligonucleotides.
Besides being able to synthesize oligonucleotides bearing xe2x80x9csensitivexe2x80x9d functionalities, the process according to this aspect of the invention can also be used in the routine synthesis of various oligonucleotides as in case of the conventional protecting groups. In addition, this new process allows for synthesis of oligonucleotides still bound to any type of solid support.
Importantly, the processes according to this aspect of the invention are compatible with and can be used in conjunction with any of the well known oligonucleotide synthetic chemistries, including the H-phosphonate, phosphoramidate and phosphotriester chemistries. Consequently, the processes according to this aspect of the invention can be used to synthesize oligonucleotides having ribonucleosides and/or primary phosphoramidate, alkylphosphonate, or methylphosphonate linkages at some internucleoside positions and other linkages at other internucleoside positions.
In a third aspect, the invention provides novel synthons for use in synthesis of oligonucleotides having base-sensitive functionalities.
One such novel synthon can be used to prepare portions of the oligonucleotide containing deoxyribonucleosides linked by any known internucleoside linkage. This monomer synthon according to the invention has the general structure III: 
wherein B is a nucleoside base, D is a 5xe2x80x2-OH blocking group (see e.g. Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 28-36 (S. Agrawal, Ed., Humana Press, 1994), preferably dimethoxytrityl or trityl, the protecting group (G) is the previously described structure I, or its preferred embodiment II, Z is hydrogen, xe2x80x94OG, xe2x80x94NG2, halogen (preferably Cl, Br or F), an xe2x80x94O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an xe2x80x94O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g, with halogen, trifluoromethyl, cyano, nitroG, acylG, acyloxyg, alkoxyG, carboxyG, carbalkoxyG; and L is a phosphoramidite, H-phosphonate, or phosphotriester leaving group, including cyclic phosphoramidite leaving groups (see Iyer et al., J. Org. Chem. 60:5388-5389 (1995)). This synthon can be used alone, with any of the known synthons, or in conjunction with any of the following synthons.
Another such novel synthon is useful in the synthesis of oligonucleotides containing ribonucleosides. This ribonucleoside synthon according to the invention has the general structure IV: 
wherein B is a nucleoside base, D is a 5xe2x80x2-OH blocking group (see e.g Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 28-36 (S. Agrawal, Ed., Humana Press, 1994), preferably dimethoxytrityl or trityl, the protecting group (G) is the previously described structure I, or its preferred embodiment II, and L is a phosphoramidite, H-phosphonate, or phosphotriester leaving group, including cyclic phosphoramidite leaving groups (see Iyer et al., J. Org. Chem. 60:5388-5389 (1995)).
Another novel synthon according to this aspect of the invention is useful for synthesizing oligonucleotides containing primary phosphoramidate internucleoside linkages. This monomer synthon according to the invention has the general structure V: 
wherein B is a nucleoside base, D is a 5xe2x80x2-OH blocking group (see e.g Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 28-36 (S. Agrawal, Ed., Humana Press, 1994), preferably dimethoxytrityl or trityl, the protecting group (G) is the previously described structure I, or its preferred embodiment II, Z is hydrogen, xe2x80x94OG, xe2x80x94NG2, halogen (preferably Cl, Br or F), an xe2x80x94O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an xe2x80x94O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, trifluoromethyl, cyano, nitroG, acylG, acyloxyG, alkoxyG, carboxyG, carbalkoxyG; and L is an H-phosphonate or H-phosphonothioate leaving group.
Another novel synthon according to this aspect of the invention is useful for synthesizing oligonucleotides containing alkylphosphonate internucleoside linkages. This monomer synthon according to the invention has the general structure VI: 
wherein B is a nucleoside base, D is a 5xe2x80x2-OH blocking group (see e.g. Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 28-36 (S. Agrawal, Ed., Humana Press, 1994), preferably dimethoxytrityl or trityl, R is an alkylphosphonamidite (preferably a methylphosphonamidite) group, Z is hydrogen, xe2x80x94OG, xe2x80x94NG2, halogen (preferably Cl, Br or F), an xe2x80x94O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an xe2x80x94O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, trifluoromethyl, cyano, nitroG, acylG, acyloxyG, alkoxyG, carboxyG, carbalkoxyG; and the protecting group (G) is the previously described structure I, or its preferred embodiment II.
In a fourth aspect, the invention provides novel oligonucleotides containing from one to about all of a variety of base-sensitive functionalities. In a preferred embodiment, such oligonucleotides may contain from one to about all ribonucleotides and/or may contain from one to about all internucleoside linkages selected from the group consisting of primary phosphoramidate and methylphosphotriester linkages. In embodiments of oligonucleotides according to this aspect of the invention that have fewer than all primary phosphoramidate or methylphosphotriester internucleoside linkages, the other internucleoside linkages may be any of the known internucleoside linkages, or may be any internucleoside linkage not yet known that can be incorporated into an oligonucleotide according to a synthetic chemistry with which the process according to the invention is compatible. Oligonucleotides containing such a mixture of internucleoside linkages are referred to herein as mixed backbone oligonucleotides. In some preferred embodiments of mixed backbone oligonucleotides according to the invention, the internucleoside linkages that are not primary phosphoramidate linkages are selected from the group consisting of phosphodiester and phosphorothioate internucleoside linkages. In some preferred embodiments of mixed backbone oligonucleotides according to the invention, several adjacent nucleosides comprising one region of the oligonucleotide are connected by primary phosphoramidate linkages, and several other adjacent nucleosides comprising another region of the oligonucleotide are connected by a different type of internucleoside linkage. These preferred oligonucleotides are referred to herein as xe2x80x9cchimericxe2x80x9d oligonucleotides.
Oligonucleotides according to the invention are useful for a variety of purposes. For example, they can be labeled with a reporter group and used as probes in conventional nucleic acid hybridization assays. They can also be used as antisense xe2x80x9cprobesxe2x80x9d of specific gene function by being used to block the expression of a specific gene in an experimental cell culture or animal system and to evaluate the effect of blocking such specific gene expression. In this use, oligonucleotides according to the invention are preferable to traditional xe2x80x9cgene knockoutxe2x80x9d approaches because they are easier to use and can be used to block specific gene expression at selected stages of development or differentiation. Finally, oligonucleotides according to the invention are useful in the antisense therapeutic approach. In this use, oligonucleotides according to the invention should have reduced polyanion-mediated side effects and improved cellular uptake.
In a fifth aspect, the invention provides methods for using oligonucleotides containing any of a variety of base-sensitive functionalities to control the expression of specific genes. Such methods comprise administering oligonucleotides according to the invention to cells or to animals, including humans. These methods may be used to assess gene function, or as a therapeutic approach to the treatment of diseases resulting from aberrant gene expression.
The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis. The patents and publications identified in this specification are within the knowledge of those skilled in this field and are hereby incorporated by reference in their entirety.
The invention provides new processes for synthesizing oligonucleotides that allow for deprotection of the oligonucleotide under more rapid and more mild conditions than existing processes. The invention further provides a nucleoside base protecting group that is stable under oligonucleotide synthesis conditions, but which can be removed under more mild conditions than existing protecting groups, as well as nucleoside synthons having such base protecting groups. In addition, the invention provides oligonucleotides containing any of various base-sensitive functionalities, and methods for using such oligonucleotides to modulate specific gene expression.
In a first aspect, the invention provides a novel nucleoside base protecting group having the general structure I: 
wherein n1, n2 and n3 are independently 0-10, wherein a, b, c, d and e are each independently hydrogen, carbon or nitrogen, and wherein the ring structure bearing substituent R3 may be aromatic or heterocyclic, wherein the nitrogen displayed is the protected amino moiety of the nucleoside base, wherein R1, R2 and R3 are independently hydrogen, or an alkyl, aryl, aralkyl, ether, hydroxy, nitrile, nitro, ester, carboxyl, or aldehyde group, and wherein dotted lines represent alternative exocyclic or endocyclic double bonds (i.e., any one of the dotted double bonds is present). In a preferred embodiment, a is hydrogen when n1 is 0 and is carbon or nitrogen when n1 is 1-10, b is hydrogen when n1, and n2 are both 0 and is carbon or nitrogen when either or both n1 and n2 are 1-10, c is hydrogen when n2 is 0 and is carbon or nitrogen when n2 is 1-10, and e is hydrogen when n3 is 0 and is carbon or nitrogen when n3 is 1-10. In a particularly preferred embodiment, compound I has n1, n2 and n3 values of 0, and a, b, c, d and e are each hydrogen, and the protecting group takes the form N-pent-4-enoyl, i.e., CH2xe2x95x90CH(CH2)2COxe2x80x94 (II). Compounds I and II protect the nucleoside base amino moieties by forming amide linkages, as in: 
where the nitrogen displayed is the protected amino moiety of the nucleoside base B.
Base protecting group I and the preferred embodiment II are particularly advantageously used because such protecting group can be removed chemoselectively by treatment with a chemoselective removing agent. Thus, in a second aspect, the invention provides processes for synthesizing oligonucleotides which allow for removal of base protecting groups under more mild conditions than existing processes. In this process, nucleoside synthons having base protecting groups according to the invention are sequentially coupled according to standard procedures to yield a base-protected oligonucleotide. The base protecting groups are then removed by a chemoselective removing agent. For purposes of the invention, a nucleoside synthon means a monomeric or multimeric nucleoside derivative appropriate for synthesis of an oligonucleotide. Preferred nucleoside synthons include monomeric nucleoside phosphoramidites, phosphotriesters, or H-phosphonates having a blocked 5xe2x80x2-OH, preferably blocked with a trityl or dimethoxytrityl group. Alternatively, for 5xe2x80x2 to 3xe2x80x2 synthesis, the 3xe2x80x2-OH will be blocked and the leaving group will be at the 5xe2x80x2 position. A chemoselective removing agent means an agent that is capable of removing a base protecting group according to the invention. In certain preferred embodiments, the chemoselective removing agent is selected from the group consisting of halogens, especially Br2, Cl2 and I2, any of which are preferably taken up in water, or in pyridine/ROH, wherein R is an alkyl, aralkyl or aryl group having 1-10 carbon atoms, or as an N-halosuccinimide. In alternative embodiments, non-chemoselective reagents may be used, such as aqueous ammonium hydroxide, alcoholic ammonia, alkali carbonates in organic solvents, primary or secondary amines, alkali hydroxides, or any amidolytic reagent, i.e., an agent capable of hydrolyzing an amide linkage.
In one preferred embodiment, deprotection and cleavage of the oligonucleotide from the support can be rapidly carried out in a single step. In this embodiment, the support-bound, protected oligonucleotide is incubated in aqueous or alcoholic ammonia or alkyl amine. Preferably, such incubation will take place for about 15 minutes to about 10 hours, more preferably from about 1 hour to about 4 hours, and most preferably from about 1 hour to about 2 hours. The preferred temperature range for such incubation is from about 20xc2x0 C. to about 70xc2x0 C., more preferably from about 50xc2x0 C. to about 60xc2x0 C., and most preferably at about 55xc2x0 C. When aqueous or alcoholic ammonia is used, the ammonia concentration is preferably from about 5% to saturated, which in the case of aquesous ammonia is about 28%. When alcoholic ammonia or alkyl amine is used, the alcohol is preferably a C1-C10 aliphatic alcohol, most preferably methanol or ethanol. When an alkyl amine is used, it is preferably a C1-C10 alkyl amine, most preferably methyl amine. This embodiment is particularly suitable for oligonucleotides having internucleoside linkages which are not labile in aqueous or alcoholic ammonia or alkyl amine, such as phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate and non-phosphate linkages.
In another preferred embodiment, neutral conditions may be employed. In this embodiment, iodine/THF/H2O or N-iodosuccinimide/THF/H2O may be used. More preferably, the protected oligonucleotide is exposed to I2 (most preferably about 0.45 M in pyridine) for a time sufficient to result in complete deprotection, most preferably for about 30 minutes or more.
In yet another preferred embodiment, anhydrous conditions may be employed to simultaneously deprotect the oligonucleotide and cleave it from the support. In this embodiment alcoholic K2CO3 is employed. Preferably the alcohol is a C1-C10 aliphatic alcohol, most preferably methanol. Incubation is preferably from about 20xc2x0 C. to about 70xc2x0, more preferably from about 20xc2x0 C. to about 30xc2x0 C., and most preferably at about 25xc2x0 C. The time for incubation is from about 15 minutes to about 6 hours, most preferably from about 3 hours to about 4 hours. The concentration of K2CO3 is preferably from about 0.005M to saturated, which in the case of K2CO3 in methanol is about 0.05 M. This embodiment is particularly attractive for synthesis of methylphosphotriester and phosphorothioate oligonucleotides.
In an additional preferred embodiment, simultaneous deprotection and cleavage from the support can be achieved through the use of NH3 in DMF. Preferably, the ammonia in DMF will be saturated. The incubation will be from about 20xc2x0 C. to about 70xc2x0 C., more preferably from about 50xc2x0 C. to about 70xc2x0 C., and most preferably at about 65xc2x0 C. This embodiment is particularly advantageous for synthesizing oligonucleotides containing primary phosphoramidate linkages, which are labile in NH4OH.
In an additional preferred embodiment, simultaneous deprotection and cleavage from the support can be achieved through the use of NH3 gas, preferably under pressure. More preferably, such deprotection is carried out at a pressure of from about 20 psi to about 200 psi, and most preferably from about 60 psi to about 120 psi. In one preferred embodiment, the deprotection reaction takes place in a glass pressure vessel, such as a Paar hydrogenation reactor, at a pressure of from about 20 psi to about 80 psi, most preferably at about 60 psi. In another preferred embodiment, the deprotection reaction can take place in a stainless steel reaction vessel, preferably at a pressure of from about 20 to about 200 psi, and most preferably from about 100 to about 120 psi. In either embodiment the reaction preferably takes place at from about 0xc2x0 C. to about 100xc2x0 C., and most preferably at about 25xc2x0 C. Under these conditions the deprotection reaction preferably proceeds from about 1 hour to about 8 hours, most preferably from about 2 hours to about 4 hours. This embodiment also is particularly advantageous for synthesizing oligonucleotides containing primary phosphoramidate linkages, which are labile in NH4OH.
The processes according to this aspect of the invention can utilize any suitable oligonucleotide synthesis chemistry in solid or solution phase, including the well known H-phophonate and phosphoramidite chemistries. In one preferred embodiment, synthesis is carried out on a suitable solid support using either H-phosphonate chemistry, phosphoramidite chemistry, or a combination of H-phosphonate chemistry and phosphoramidite chemistry (i.e., H-phosphonate chemistry for some cycles and phosphoramidite chemistry for other cycles). Suitable solid supports include any of the standard solid supports used for solid phase oligonucleotide synthesis, such as controlled-pore glass (CPG) or polymer supports. (See, e.g., Pon, Methods in Molec. Biol. 20: 465 (1993)). Synthesis on such a solid support begins with coupling a nucleoside synthon according to the invention to a nucleoside that is covalently linked the solid support (i.e., linked to a functionality on the solid support, preferably an amino or hydroxyl functionality). More generally, the processes according to this aspect of the invention can be used with any of the chemistries commonly used for oligonucleotide synthesis, whether in solution phase or in solid phase. Thus, in one preferred embodiment, the invention provides a process for synthesizing an oligonucleotide, such process comprising coupling a suitable nucleoside synthon, such as a nucleoside H-phosphonate, a nucleoside phosphoramidite, or a nucleoside phosphotriester to a nucleoside and deprotecting a nucleoside base with a reagent comprising (1) a halogen in water, in an ethereal solvent such as ether or THF, or in pyridine/ROH, wherein R is an alkyl, aralkyl or aryl group having 1-10 carbon atoms, or (2) a suitable halide releasing reagent, such as N-halosuccinimide sodium hypochlorite, or N-iodosuccinimide in paratoluenesulfonic acid. The nucleoside to which the nucleoside synthon is coupled may be a monomer, a multimer, or it may be the terminal nucleoside of a growing oligonucleotide chain. In either case, the nucleoside or growing oligonucleotide chain may be support-bound or free in solution.
The use of this new process provides numerous advantages. For example the process""s mild procedure for removing the protecting group without affecting the integrity of other functionalities present in the oligonucleotide makes it possible to prepare novel analogs of oligonucleotides such as ribonucleoside-containing oligonucleotides, alkylphosphotriesters, certain base-sensitive phosphoramidate and other base-sensitive oligonucleotides. Surprisingly, the process according to the invention is more efficient than existing processes. This is believed to be due in part to the greater purity of the novel synthons according to the invention, relative to existing synthons.
One preferred use of this aspect of the invention is in the synthesis of oligonucleotides containing from one to about all ribonucleosides. Preferably, such synthesis employs a phosphoramidite, H-phosphonate or phosphotriester nucleoside monomer synthon having novel protecting groups according to the invention on the nucleoside base, as well as on the 2xe2x80x2 hydroxyl of the nucleoside sugar.
Another preferred use of this aspect of the invention is in the synthesis of an oligonucleotide containing from one to about all primary phosphoramidate internucleoside linkages. The primary phosphoramidate internucleoside linkage has the structure: 
wherein xe2x80x9cNuclxe2x80x9d represents the 3xe2x80x2 position of a first nucleoside, xe2x80x9cNuc2xe2x80x9d represents the 5xe2x80x2 position of a second nucleoside, and X represents S or O. This process comprises condensing a nucleoside H-phosphonate with another nucleoside, wherein at least one of the nucleosides has a nucleoside base-protecting group according to the invention, to produce adjacent nucleosides coupled by an H-phosphonate internucleoside linkage, wherein at least one of the nucleosides has a nucleoside base-protecting group according to the invention, aminating the H-phosphonate internucleoside linkage to produce a primary phosphoramidate linkage, and chemoselectively removing the nucleoside base-protecting group without cleaving the primary phosphoramidate linkage. This process allows for synthesis, for the first time, of oligonucleotides containing primary phosphoramidate internucleoside linkages.
Another preferred use of this aspect of the invention is in the synthesis of oligonucleotides containing from one to about all alkylphosphonate internucleoside linkages, most preferably methylphosphonate internucleoside linkages. This new process comprises sequentially coupling nucleoside alkylphosphonamidite (preferably methylphosphonamidite) synthons having base protecting groups according to the invention to produce a base-protected oligonucleotide having an alkylphosphonate internucleoside linkage, followed by deprotection using a chemoselective removing agent. In one preferred embodiment, this aspect of the invention comprises coupling together an alkylphosphonamidite nucleoside synthon, most preferably a methylphosphonamidite nucleoside synthon, with a nucleoside or oligonucleoside having a free 5xe2x80x2 hydroxyl group to produce a base-protected oligonucleotide having an alkylphosphonite (III) internucleoside linkage having as a bridging oxygen the oxygen from the free 5xe2x80x2 hydroxyl group from the nucleoside or nucleotide, oxidizing the internucleoside linkage to an alkylphosphonate linkage, and deprotecting the oligonucleotide using a chemoselective removing agent. As used herein, the terms xe2x80x9cnucleosidexe2x80x9d or xe2x80x9coligonucleosidexe2x80x9d include those having appropriately protected reactive functionalities, either in accordance with the present invention or with conventional protecting groups known in the art (see e.g Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 1-36 (S. Agrawal, Ed., Humana Press, 1994). Preferably, the oxidation of the internucleoside linkage to an alkylphosphonate linkage utilizes a phosphite oxidizing agent such as tert-butyl hydroperoxide or other well known agents (see Beaucage and Iyer, Tetrahedron 48: 2223-2311 (1992)).
Another preferred use of this aspect of the invention is in the synthesis of oligonucleotides containing from one to about all methylphosphotriester internucleoside linkages. The methylphosphotriester internucleoside linkage has the structure 
wherein xe2x80x9cNuc1xe2x80x9d represents the 3xe2x80x2 position of a first nucleoside, xe2x80x9cNuc2xe2x80x9d represents the 5xe2x80x2 position of a second nucleoside, and X is sulfur or oxygen.
One embodiment of this new process comprises condensing in the presence of 1H-tetrazole a methoxy-3xe2x80x2-O-(phosphoramidite)-5xe2x80x2-O-(4,4xe2x80x2-dimethoxytriphenyl)methyl nucleoside with another nucleoside, wherein at least one of the nucleosides has a nucleoside base-protecting group, to produce adjacent nucleosides coupled by a phosphite linkage, wherein at least one of the nucleosides has a nucleoside base-protecting group, oxidizing the internucleotidic phosphite linkage to yield an O-methylphosphotriester or O-methylphosphorothioate linkage, and chemoselectively removing the nucleoside base-protecting group without demethylating the O-methylphosphotriester or O-methylphophorothioate linkage(s). Another embodiment comprises condensing in the presence of a suitable activator, such as pivaloyl chloride, a nucleoside H-phosphonate or thio-H-phosphonate with another nucleoside, wherein at least at least one of the nucleosides has a nucleoside base protecting group, to produce adjacent nucleosides coupled by an H-phosphonate or thio-H-phosphonate linkage, wherein at least one of the nucleosides has a nucleoside base protecting group, oxidizing the H-phosphonate linkage in carbon tetrachloride/pyridine/methanol to produce an O-methylphosphotriester or O-methylphosphorothioate linkage, then chemoselectively removing the nucleoside base protecting group without memethylating the O-methylphosphotriester or O-methylphosphorothioate linkage, as described previously, and most preferably in I2/pyridine/methanol.
The versatility of chemical synthetic approach of the processes according to this aspect of the invention makes such processes suitable for the synthesis of any of a broad class of compounds, all of which are referred to herein as xe2x80x9coligonucleotidesxe2x80x9d. For purposes of the invention, the term oligonucleotide includes polymers of two or more deoxyribonucleotide, or any modified nucleoside, including 2xe2x80x2-halo-nucleosides, 2xe2x80x2O-substituted ribonucleosides, deazanucleosides or any combination thereof. Such monomers may be coupled to each other by any of the numerous known internucleoside linkages. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof. The term oligonucleotide also encompasses such polymers having chemically modified bases or sugars and/or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines and adamantane. For purposes of the invention the term xe2x80x9c2xe2x80x2-O-substitutedxe2x80x9d means substitution of the 2xe2x80x2 position of the pentose moiety with a halogen (preferably Cl, Br, or F), or an xe2x80x94O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an xe2x80x94O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or such 2xe2x80x2 substitution may be with a hydroxy group (to produce a ribonucleoside), an amino or a halo group, but not with a 2xe2x80x2-H group.
The use of this new process provides numerous advantages. For example the process""s chemoselective capacity for removing the protecting group without affecting the integrity of other functionalities present in the oligonucleotide makes it possible to prepare novel analogs of oligonucleotides such as oligoribonucleotides, alkylphosphotriesters, certain base sensitive phosphoramidates and other base sensitive oligonucleotides. Besides being able to synthesize oligonucleotides bearing xe2x80x9csensitivexe2x80x9d functionalities, it can also be used in the routine synthesis of various oligonucleotides as in case of the conventional protecting groups. In addition, this new process allows for synthesis of oligonucleotides still bound to any type of solid support. Where an unprotected, support bound oligonucleotide is desired, the full length support-bound oligonucleotide will have its internucleoside linkages oxidized, followed by contacting the oligonucleotide with a chemoselective removing agent to cleave the base protecting group. In the phosphoramidite approach, this is followed by treatment with anhydrous triethylamine to cleave the beta-cyanoethyl moiety.
Additionally, according to this aspect of the invention, support-bound branched oligonucleotides can be synthesized using, for example glycol residues in which one hydroxyl group is protected by e.g., DMT, and the other by a protecting group according to the invention. Then the DMT group may be selectively removed and an oligonucleotide synthesized from the resulting unprotected hydroxyl. Upon completion of that oligonucleotide, the hydroxyl moiety protected by the protecting group according to the invention can be deprotected with a chemoselective removing agent and another, different oligonucleotide synthesized from it.
In a third aspect, the invention provides novel synthons for use in synthesis of oligonucleotides having base-sensitive functionalities. One such novel synthon can be used to prepare portions of the oligonucleotide containing deoxyribonucleosides linked by any known internucleoside linkage. This monomer synthon according to the invention has the general structure III: 
wherein B is a nucleoside base, D is a 5xe2x80x2-OH blocking group (see e.g. Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 28-36 (S. Agrawal, Ed., Humana Press, 1994), preferably dimethoxytrityl or trityl, the protecting group (G) is the previously described structure I, or its preferred embodiment II, Z is hydrogen, xe2x80x94OG, xe2x80x94NG2, halogen (preferably Cl, Br or F), an xe2x80x94O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an xe2x80x94O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, trifluoromethyl, cyano, nitroG, acylg, acyloxyG, alkoxyG, carboxyG, carbalkoxyG; and L is a phosphoramidite, H-phosphonate, or phosphotriester leaving group, including cyclic phosphoramidite leaving groups (see Iyer et al., J. Org. Chem. 60:5388-5389 (1995)). This synthon can be used alone, with any of the known synthons, or in conjunction with any of the following synthons. In an alternative embodiment, D can be a 3xe2x80x2-OH blocking group and L can be a 3xe2x80x2 leaving group, for synthesis in the 5xe2x80x2 to 3xe2x80x2 direction. In another embodiment, the novel synthon can be a nucleoside dimer or multimer.
Another such novel synthon is useful in the synthesis of oligonucleotides containing ribonucleosides. This monomer synthon according to the invention has the general structure IV: 
wherein B is a nucleoside base, D is a 5xe2x80x2-OH blocking group (see, e.g. Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 28-36 (S. Agrawal, Ed., Humana Press, 1994), preferably dimethoxytrityl or trityl, the protecting group (G) is the previously described structure I, or its preferred embodiment II, and L is a phosphoramidite, H-phosphonate, or phosphotriester leaving group, including cyclic phosphoramidite leaving groups (see Iyer et al., J. Org. Chem. 60:5388-5389 (1995)).
A scheme for synthesis of such a monomer having a particularly preferred embodiment of the protecting group according to the invention is shown in FIG. 1. According to this scheme, the monomer synthon is synthesized from the ribonucleoside by first protecting the 3xe2x80x2 and 5xe2x80x2 hydroxyl groups as the cyclic silyl ether derivative using the Markiewicz reagent. Then the N-pent-4-enoyl (PNT) group is installed at the nucleobase and the 2xe2x80x2 hydroxyl of the ribose unit using PNT anhydride or using pent-4-enoic acid in the presence of dicyclohexylcarbodiimide (DCC). The 3xe2x80x2 and 5xe2x80x2 protecting groups are removed using tetrabutylammonium fluoride, followed by conversion of the diol to the 5xe2x80x2-O-4,4-dimethoxytrityl 3xe2x80x2-O-phosphoramidite monomer synthon by adaptation of standard phosphoramidite synthesis protocols using the appropriate chlorophosphitylation reagent. A minor isomerization product resulting from 2xe2x80x2-3xe2x80x2 migration of groups is also formed, but is readily removed by chromatography. The formation of this minor product can also be substantially reduced by using bis-N,N-diisopropylphosphoramidite as the phosphitylating reagent.
Another novel synthon according to this aspect of the invention is useful for synthesizing oligonucleotides containing primary phosphoramidate internucleoside linkages. This monomer synthon according to the invention has the general structure V: 
wherein B is a nucleoside base, D is a 5xe2x80x2-OH blocking group (see e.g Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 28-36 (S. Agrawal, Ed., Humana Press, 1994), preferably dimethoxytrityl or trityl, the protecting group (G) is the previously described structure I, or its preferred embodiment II, Z is hydrogen, xe2x80x94OG, xe2x80x94NG2, halogen (preferably Cl, Br or F), an xe2x80x94O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an xe2x80x94O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, trifluoromethyl, cyano, nitroG, acylg, acyloxyG, alkoxyG, carboxyG, carbalkoxyG; and L is an H-phosphonate or H-phosphonothioate (see Kers et al., Nucleosides and Nucleotides 15: 361-378 (1996)) leaving group.
Another novel synthon according to this aspect of the invention is useful for synthesizing oligonucleotides containing alkylphosphonate internucleoside linkages. This monomer synthon according to the invention has the general structure VI: 
wherein B is a nucleoside base, D is a 5xe2x80x2-OH blocking group (see e.g. Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 28-36 (S. Agrawal, Ed., Humana Press, 1994), preferably dimethoxytrityl or trityl, R is an alkylphosphonamidite (preferably a methylphosphonamidite) group, Z is hydrogen, xe2x80x94OG, xe2x80x94NG2, halogen (preferably Cl, Br, or F), an xe2x80x94O-lower alkyl group containing 1-6 atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, trifluoromethyl, cyano, nitroG, acylG, acyloxyG, alkoxyG, carboxyG, carbalkoxyG; and the protecting group (G) is the previously described structure I, or its preferred embodiment II.
For purposes of this aspect of the invention, the term xe2x80x9calkyl groupxe2x80x9d means a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or an allyl group having 2-6 carbon atoms, wherein such alkyl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups.
In a fourth aspect, the invention provides novel oligonucleotides containing from one to about all of a variety of base-sensitive functionalities. In a preferred embodiment, such oligonucleotides may contain from one to about all ribonucleotides and/or may contain from one to about all internucleoside linkages selected from the group consisting of primary phosphoramidate and methylphosphotriester linkages. In addition, oligonucleosides according to this aspect of the invention may have alkylphosphonate or alkylphosphonite (III) internucleoside linkages. In embodiments of oligonucleotides according to this aspect of the invention that have fewer than all primary phosphoramidate or methylphosphotriester internucleoside linkages, the other internucleoside linkages may be any of the known internucleoside linkages, or may be any internucleoside linkage not yet known that can be incorporated into an oligonucleotide according to a synthetic chemistry with which the process according to the invention is compatible. In certain preferred embodiments, the other internucleoside linkages are phosphodiester or phosphorothioate linkages. In the case of phosphorothioate internucleoside linkages, the linkages may be phosphorothioate mixed enantiomers or stereoregular phosphorothioates(see Iyer et al., Tetrahedron Asymmetry 6: 1051-1054 (1995).
Oligonucleotides containing such a mixture of internucleoside linkages are referred to herein as mixed backbone oligonucleotides. In some preferred embodiments of mixed backbone oligonucleotides according to the invention, the internucleoside linkages that are not primary phosphoramidate or methylphosphotriester linkages are selected from the group consisting of phosphodiester and phosphorothioate internucleoside linkages. In some preferred embodiments of mixed backbone oligonucleotides according to the invention, several adjacent nucleosides comprising one region of the oligonucleotide are connected by primary phosphoramidate or methylphosphotriester linkages, and several other adjacent nucleosides comprising another region of the oligonucleotide are connected by a different type of internucleoside linkage. These preferred oligonucleotides are referred to herein as xe2x80x9cchimericxe2x80x9d oligonucleotides. In certain particularly preferred chimeric oligonucleotides according to the invention, the oligonucleotide comprises a primary phosphoramidate or methylphosphotriester region and a phosphorothioate and/or phosphodiester and/or alkylphosphonate region. In this context, a xe2x80x9cprimary phosphoramidate regionxe2x80x9d or a xe2x80x9cmethylphosphotriester regionxe2x80x9d is a region within an oligonucleotide of from about 2 to about 15 contiguous nucleosides linked to each other through primary phosphoramidate or methylphosphotriester linkages according to the invention, respectively. A xe2x80x9cphosphorothioate regionxe2x80x9d is a region within an oligonucleotide of from about 4 to about 20 contiguous nucleosides linked to each other through phosphorothioate linkages. A xe2x80x9cphosphodiester regionxe2x80x9d is a region within an oligonucleotide of from about 4 to about 20 contiguous nucleosides linked to each other through phosphodiester linkages. An xe2x80x9calkylphosphonate regionxe2x80x9d is a region within an oligonucleotide of from about 4 to about 20 contiguous nucleosides linked to each other through alkylphosphonate, preferably methylphosphonate linkages. In most preferred chimeric oligonucleotides according to the invention, the oligonucleotide comprises a phosphorothioate or phosphodiester region flanked on either side by a primary phosphoramidate or methylphosphotriester region, or alternatively, a primary phosphoramidate or methylphosphotriester region flanked on either side by a phosphorothioate or phosphodiester region. In one preferred embodiment the nucleosides of the primary phosphoramidate or methylphosphotriester region are ribonucleosides or 2xe2x80x2-O-substituted ribonucleotides, as defined above herein. Preferred chimeric oligonucleotides according to the invention are further characterized by having the ability to activate RNaseH.
Preferably, such oligonucleotides will have from about 12 to about 50 nucleotides, most preferably from about 17 to about 35 nucleotides. Preferably, such oligonucleotides will have a nucleotide sequence that is complementary to a genomic region, a gene, or an RNA transcript thereof. The term complementary means having the ability to hybridize to a genomic region, a gene, or an RNA transcript thereof under physiological conditions. Such hybridization is ordinarily the result of base-specific hydrogen bonding between complementary strands, preferably to form Watson-Crick or Hoogsteen base pairs, although other modes of hydrogen bonding, as well as base stacking can also lead to hybridization. As a practical matter, such hybridization can be inferred from the observation of specific gene expression inhibition. The gene sequence or RNA transcript sequence to which the modified oligonucleotide sequence is complementary will depend upon the biological effect that is sought to be modified. In some cases, the genomic region, gene, or RNA transcript thereof may be from a virus. Preferred viruses include, without limitation, human immunodeficiency virus (type 1 or 2), influenza virus, herpes simplex virus (type 1 or 2), Epstein-Barr virus, cytomegalovirus, respiratory syncytial virus, influenza virus, hepatitis B virus, hepatitis C virus and papilloma virus. In other cases, the genomic region, gene, or RNA transcript thereof may be from endogenous mammalian (including human) chromosomal DNA. Preferred examples of such genomic regions, genes or RNA transcripts thereof include, without limitation, sequences encoding vascular endothelial growth factor (VEGF), beta amyloid, DNA methyltransferase, protein kinase A, ApoE4 protein, p-glycoprotein, c-MYC protein, BCL-2 protein, protein kinase A and CAPL. In yet other cases, the genomic region, gene, or RNA transcript thereof may be from a eukaryotic or prokaryotic pathogen including, without limitation, Plasmodium falciparum, Plasmodium malarie, Plasmodium ovale, Schistosoma spp., and Mycobacterium tuberculosis. 
In a fifth aspect, the invention provides methods for using oligonucleotides containing any of a variety of base-sensitive functionalities to control the expression of specific genes. Such methods comprise administering oligonucleotides according to the invention to cells or to animals, including humans. These methods may be used to assess gene function, or as a therapeutic approach to the treatment of diseases resulting from aberrant gene expression.
Due to the nonionic character of certain preferred base labile functionalities in oligonucleotides according to the invention, the invention further provides a method for therapeutically treating, with reduced side effects, a disease caused by aberrant gene expression, the method comprising administering to an individual having the disease a composition of matter comprising an oligonucleotide according to the invention, wherein the oligonucleotide is complementary to a gene that is aberrantly expressed, wherein such aberrant expression causes the disease. In this context, aberrant gene expression means expression in a host organism of a gene required for the propagation of a virus or a prokaryotic or eukaryotic pathogen, or inappropriate expression of a host cellular gene. Inappropriate host cellular gene expression includes expression of a mutant allele of a cellular gene, or underexpression or overexpression of a normal allele of a cellular gene, such that disease results from such inappropriate host cellular gene expression. Preferably, such administration should be parenteral, oral, sublingual, transdermal, topical, intranasal or intrarectal. Administration of the therapeutic compositions can be carried out using known procedures at dosages and for periods of time effective to reduce symptoms or surrogate markers of the disease. When administered systemically, the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of oligonucleotide from about 0.01 micromolar to about 10 micromolar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, a total dosage of oligonucleotide will range from about 0.1 mg oligonucleotide per patient per day to about 200 mg oligonucleotide per kg body weight per day. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode. In a preferred embodiment, after the composition of matter is administered, one or more measurement is taken of biological effects selected from the group consisting of complement activation, mitogenesis and inhibition of thrombin clot formation.